The control of mRNA stability has recently emerged as a critical yet poorly understood determinant in the regulation of eukaryotic gene expression. Changes of mRNA stability can have profound consequences that may become manifest as clinical phenotypes. Malignancies that arise from aberrant expression of proto-oncogenes, and thalassemias, resulting from improper globin gene expression, are two examples indicating the importance of proper regulation of mRNA stability. Yet very little is known concerning the components that control mammalian mRNA turnover. We have devised an in vitro mRNA decay assay that recapitulates regulated mRNA turnover observed in cells and have shown that mammalian mRNA can be degraded from either the 5' or the 3' end. We have identified the two mammalian decapping enzymes, one of which is a homolog of the yeast Dcp2 protein and functions on capped mRNA. The second is a scavenger decapping activity, DcpS, which functions on the residual cap structure resulting from 3' to 5' decay of an mRNA. We have demonstrated human Dcp2 (hDcp2) is an RNA-binding protein whose activity is regulated by both the poly(A) tail and a potent inhibitor protein implicated in mental retardation. We have also observed that the human DcpS, in addition to its role in hydrolyzing the cap structure following mRNA decay, functions in a novel regulatory role to facilitate mRNA decay. The long term objective of this proposal is to understand the determinants and nucleases that regulate mRNA decay in mammals. The focus of this proposal is to: (AIM 1) investigate the regulatory mechanisms that control hDcp2 decapping; (AIM 2) determine the mechanism by which DcpS decapping enzyme functions to regulate mRNA decay; and (AIM 3) determine the contribution of the different pathways of mRNA decay involving the two decapping enzymes in vivo. This work will provide significant insights into a fundamental mechanism involved in the post transcriptional control of gene expression, that of mRNA turnover, and will provide a framework for novel approaches to regulate gene expression for therapeutic intervention.